RESUMEN
Proteases derived from Streptomyces demonstrate numerous commendable properties, rendering it extensively applicable in biotechnology and various industrial sectors. This study focused on the purification and characterization of the thermostable protease obtained from Streptomyces sp. CNXK100. The purified protease exhibited an estimated molecular weight of 27 kDa, with optimal activity at 75°C and pH 8.0. Notably, the enzyme remained active even without any metal ions and fully active in the presence of Na+, K+, Mg2+, and Cu2+metal ions. The kinetic parameters were determined with a KM value of 3.13 mg/ml and a Vmax value of 3.28 × 106 U/mg. Furthermore, the protease has demonstrated notable stability when subjected to a treatment temperature of up to 65°C for 60 minutes, and across a broad pH range extending from 5.0 to 10.0. This protease also demonstrated resilience against a spectrum of harsh conditions, including exposure to organic solvents, surfactants, bleaching agents, and proteolytic enzymes. Additionally, the enzyme maintained its activity following treatment with commercial detergents, accomplishing complete thrombus lysis at a concentration of 2.50 mg/ml within 4 hours. Remarkably, the protease exhibited stability in terms of activity and protein concentration for 70 days at 4°C. These findings underscore the potential industrial applications of the thermostable protease from Streptomyces sp. CNXK100.
Asunto(s)
Proteínas Bacterianas , Estabilidad de Enzimas , Péptido Hidrolasas , Streptomyces , Temperatura , Streptomyces/enzimología , Streptomyces/química , Concentración de Iones de Hidrógeno , Cinética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/aislamiento & purificación , Peso Molecular , Metales/farmacología , Metales/químicaRESUMEN
Rapamycin is a clinically important macrolide agent with immunosuppressant and antiproliferative properties, produced by the actinobacterium, Streptomyces rapamycinicus. Two cytochrome P450 enzymes are involved in the biosynthesis of rapamycin. CYP107G1 and CYP122A2 catalyze the oxidation reactions of C27 and C9 of pre-rapamycin, respectively. To understand the structural and biochemical features of P450 enzymes in rapamycin biosynthesis, the CYP107G1 and CYP122A2 genes were cloned, their recombinant proteins were expressed in Escherichia coli, and the purified enzymes were characterized. Both enzymes displayed low spin states in the absolute spectra of ferric forms, and the titrations with rapamycin induced type I spectral changes with Kd values of 4.4 ± 0.4 and 3.0 ± 0.3 µM for CYP107G1 and CYP122A2, respectively. The X-ray crystal structures of CYP107G1 and its co-crystal complex with everolimus, a clinical rapamycin derivative, were determined at resolutions of 2.9 and 3.0 Å, respectively. The overall structure of CYP107G1 adopts the canonical scaffold of cytochrome P450 and possesses large substrate pocket. The distal face of the heme group is exposed to solvents to accommodate macrolide access. When the structure of the everolimus-bound CYP107G1 complex (CYP107G1-Eve) was compared to that of the ligand-free CYP107G1 form, no significant conformational change was observed. Hence, CYP107G1 has a relatively rigid structure with versatile loops to accommodate a bulky substrate. The everolimus molecule is bound to the substrate-binding pocket in the shape of a squeezed donut, and its elongated structure is bound perpendicular to a planar heme plane and I-helix.
Asunto(s)
Proteínas Bacterianas/química , Sistema Enzimático del Citocromo P-450/química , Streptomyces/enzimología , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/genética , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas Recombinantes , Sirolimus/metabolismo , Streptomyces/genéticaRESUMEN
Streptomyces avermitilis is an actinobacterium known to produce clinically useful macrolides including avermectins. CYP107L2 from S. avermitilis shares a high sequence similarity with the PikC (CYP107L1) from S. venezuelae. To elucidate the structural features of CYP107L2, we conducted biochemical and structural characterization of CYP107L2 from S. avermitilis. The CYP107L2 gene was cloned, and its recombinant protein was expressed and purified. The CYP107L2 showed a low-spin state of heme, and the reduced form yielded the CO difference spectra with a maximal absorption at 449 nm. Binding of pikromycin and lauric acid yielded the typical type I spectra with Kd values of 4.8 ± 0.3 and 111 ± 9 µM, respectively. However, no metabolic product was observed in the enzyme reaction. X-ray crystal structures of the ligand-free CYP107L2 and its complex with lauric acid were determined at the resolution of 2.6 and 2.5 Å, respectively. CYP107L2 showed a well-conserved CYP structure with a wide-open substrate-binding cavity. The lauric acid is bound mainly via hydrophobic interactions with the carboxylate group of lauric acid coordinated to the heme of P450. Glu-40 and Leu-382 residues in the CYP107L2 complex with lauric acid showed significant conformational changes to provide plentiful room for the lauric acid in the substrate-binding site.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Láuricos/metabolismo , Streptomyces/enzimología , Sitios de Unión , Cristalografía por Rayos X , Macrólidos/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , Streptomyces/química , Streptomyces/metabolismoRESUMEN
CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of 2.6 Å. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant's catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Oligomicinas/metabolismo , Streptomyces/metabolismo , Triptófano/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Mutación , Oligomicinas/química , Unión Proteica , Estructura Secundaria de Proteína , Streptomyces/química , Triptófano/metabolismoRESUMEN
Streptomyces avermitilis contains 33 cytochrome P450 genes in its genome, many of which play important roles in the biosynthesis process of antimicrobial agents. Here, we characterized the biochemical function and structure of CYP107W1 from S. avermitilis, which is responsible for the 12-hydroxylation reaction of oligomycin C. CYP107W1 was expressed and purified from Escherichia coli. Purified proteins exhibited the typical CO-binding spectrum of P450. Interaction of oligomycin C and oligomycin A (12-hydroxylated oligomycin C) with purified CYP107W1 resulted in a type I binding with Kd values of 14.4 ± 0.7 µM and 2.0 ± 0.1 µM, respectively. LC-mass spectrometry analysis showed that CYP107W1 produced oligomycin A by regioselectively hydroxylating C12 of oligomycin C. Steady-state kinetic analysis yielded a kcat value of 0.2 min(-1) and a Km value of 18 µM. The crystal structure of CYP107W1 was determined at 2.1 Å resolution. The overall P450 folding conformations are well conserved, and the open access binding pocket for the large macrolide oligomycin C was observed above the distal side of heme. This study of CYP107W1 can help a better understanding of clinically important P450 enzymes as well as their optimization and engineering for synthesizing novel antibacterial agents and other pharmaceutically important compounds.
Asunto(s)
Antibacterianos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Oligomicinas/biosíntesis , Streptomyces/metabolismo , Antibacterianos/química , Secuencia de Bases , Cristalización , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Cartilla de ADN , Modelos Moleculares , Oligomicinas/química , Reacción en Cadena de la Polimerasa , Streptomyces/enzimologíaRESUMEN
Along with the co-chaperonin GroES, the chaperonin GroEL plays an essential role in enhancing protein folding or refolding and in protecting proteins against misfolding and aggregation in the cellular environment. The XoGroEL gene (XOO_4288) from Xanthomonas oryzae pv. oryzae was cloned and the protein was expressed, purified and crystallized. The purified XoGroEL protein was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to a resolution of 3.4 Å. The crystal belonged to the orthorhombic space group P212121 with 14 monomers in the asymmetric unit, with a corresponding VM of 2.7 Å(3) Da(-1) and a solvent content of 54.5%.
Asunto(s)
Proteínas Bacterianas/química , Chaperoninas/química , Xanthomonas , Proteínas Bacterianas/aislamiento & purificación , Chaperoninas/aislamiento & purificación , Cristalización , Cristalografía por Rayos XRESUMEN
Acinetobacter baumannii causes bacteraemia, pneumonia, other respiratory-tract and urinary-tract infections in humans. OXA-23 carbapenemase-producing A. baumannii K0420859 (A. baumannii OXA-23) is resistant to carbapenem, a common antibacterial drug. To develop an efficient and novel antibacterial drug against A. baumannii OXA-23, D-alanine-D-alanine ligase, which is essential in bacterial cell-wall synthesis, is of interest. Here, the D-alanine-D-alanine ligase (AbDdl) gene from A. baumannii OXA-23 was cloned and expressed, and the AbDdl protein was purified and crystallized; this enzyme can be used as a novel target for an antibacterial drug against A. baumannii OXA-23. The AbDdl crystal diffracted to a resolution of 2.8â Å and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 113.4, b = 116.7, c = 176.5â Å, a corresponding VM of 2.8â Å(3)â Da(-1) and a solvent content of 56.3%, and six protomers in the asymmetric unit.
Asunto(s)
Acinetobacter baumannii/enzimología , Cristalización/métodos , Cristalografía por Rayos X/métodos , Péptido Sintasas/química , Péptido Sintasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , beta-Lactamasas/metabolismo , Péptido Sintasas/genética , Proteínas Recombinantes/genéticaRESUMEN
Campylobacter jejuni is one of the major foodborne pathogens causing human infection. Peptide deformylase, a metallohydrolase, catalyzes the deformylation of N-formylated methionine in newly synthesized polypeptides in prokaryotes and some eukaryotic organelles. The deformylation process is an essential step in protein synthesis and has attracted much attention as a potential target for the development of novel antibacterial agents. Here, the cloned codon-optimized def gene from C. jejuni was synthesized and the protein was expressed, purified and crystallized. C. jejuni peptide deformylase crystals obtained at pH 7.0 and pH 6.5 diffracted to 2.9â Å resolution and belonged to the trigonal space group R3, with unit-cell parameters a=b=105.7, c=58.0â Å. One monomer existed in the asymmetric unit, with a corresponding VM of 3.1â Å3â Da(-1) and a solvent content of 60.4%.
Asunto(s)
Amidohidrolasas/química , Campylobacter jejuni/enzimología , Amidohidrolasas/genética , Campylobacter jejuni/genética , Cristalización , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , HumanosRESUMEN
Cellobiose 2-epimerase epimerizes and isomerizes ß-1,4- and α-1,4-gluco-oligosaccharides. N-Acyl-D-glucosamine 2-epimerase (DT_epimerase) from Dictyoglomus turgidum has an unusually high catalytic activity towards its substrate cellobiose. DT_epimerase was expressed, purified and crystallized. Crystals were obtained of both His-tagged DT_epimerase and untagged DT_epimerase. The crystals of His-tagged DT_epimerase diffracted to 2.6â Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a=63.9, b=85.1, c=79.8â Å, ß=110.8°. With a Matthews coefficient VM of 2.18â Å3â Da(-1), two protomers were expected to be present in the asymmetric unit with a solvent content of 43.74%. The crystals of untagged DT_epimerase diffracted to 1.85â Å resolution and belonged to the orthorhombic space group P212121, with unit-cell parameters a=55.9, b=80.0, c=93.7â Å. One protomer in the asymmetric unit was expected, with a corresponding VM of 2.26â Å3â Da(-1) and a solvent content of 45.6%.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/química , Carbohidrato Epimerasas/química , Proteínas Portadoras/química , Cristalización , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Histidina , OligopéptidosRESUMEN
Acinetobacter baumannii has received much attention owing to its exceptional ability to develop resistance to currently available antibiotics. Alanine racemase (ALR) catalyzes the racemization of L-alanine to D-alanine with pyridoxal 5'-phosphate (PLP) as a cofactor. The D-alanine product is an essential component of the bacterial cell wall and ALR is a potential target for the development of novel antibacterial drugs. The alr gene from A. baumannii was cloned and the protein (AbALR) was expressed, purified and crystallized. The AbALR crystal diffracted to 2.3â Å resolution and belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 55.1, b = 85.0, c = 167.7â Å. Two protomers were present in the asymmetric unit, with a corresponding V(M) value of 2.3â Å(3)â Da(-1) and a solvent content of 47.5%.